Oils and fats are triglycerides which typically consist of glycerol and saturated and unsaturated fatty acids. These are being increasingly used in recent times for the development of competitive, powerful products, which are both consumer-friendly and environment-friendly (Hill K, Pure and Applied Chemistry 72 (2000) pp. 1255-1264). For most of the further uses, oils and fats must be split into the so-called oleochemical base materials, predominantly fatty acids and glycerol. Intermediates as well as monoacyl glycerols (MAG's), diacylglycerols (DAG's), fatty acid methyl esters and also hydrogenation products of the fatty acid methyl esters i.e. fatty alcohols find immense use in the oleochemical industry (Falbe et al., Angew. Chem. Int. Ed. Engl., 27 (1988) pp. 41-62).
The hydrolysis of triacylglycerols (TAG) to yield free fatty acids (FAs), MAGs and glycerol is the primary reaction, the fatty acids thus produced are further interesterified, transesterified, or are converted into high-value fatty alcohols. These base materials are then used as intermediates in production of washing and cleansing agents, cosmetics, surfactants, polymers and lubricants. There are many useful mono-glycerides of immense commercial interest like glycerol monostearates, monooleates and monoricinoleates that are produced synthetically from fatty acids and glycerol to the tune of more than 10,000 tons annually.
Hydrolysis of oil has been accomplished commercially by using catalysts at high temperature and high pressure like Twitchell process and Colgate-Emery process. Colour development, formation of by-products, induction of polymerization and requirement of subsequent distillation are major drawbacks of these processes. The reaction by-products are associated with undesired dark colour and burnt taste, and thus need specialized techniques (e.g. molecular distillation) to remove colour and by-products. The rapid advances in the field have led to the introduction of milder chemical reaction conditions for fat-splitting; however, the process is still very high on CAPEX and calls for better technologies.
Hydrolysis of oils or fats, specifically with lipase as biocatalyst, provides several advantages including reaction at atmospheric pressure and low temperatures. There are several additional advantages of the enzymatic process in addition to the possibility of controlling the reaction to give MAGs. However, till date fat splitting through the use of lipolytic enzymes has been carried out only in experimental trials. Enzymatic process has never been commercialized due to high cost and long reaction times.
Hammond et al. (Journal of American Oil Chemist's Society, 67 (1990), pp. 761-765) describe 90% lipolysis in about 58 days where, only 10% conversion was achieved in 4 days. The authors postulate that the slow rate of hydrolysis may be due to inhibition of the enzyme by glycerol, a product of the reaction. U.S. Pat. No. 5,932,458 describes use of lipase catalysts recovered from pulverised seeds for splitting of fats and oils of various types, differing in the degree of saturation or hydroxylation.
Microbial lipase has also been studied as catalyst of hydrolysis of sunflower oil, soybean lecithin and their mixtures at 60° C. in a biphasic mixture heptane-buffer pH 7.0 (Ferreira et al., Enzyme and Microbial Technology, 41(1-2) 2007, pp. 35-43). Hydrolysis of palm oil with an yield of 32-50% of MAGs using membrane bound lipase in a two-phase reaction system (Tianwei Tan et al., Journal of Molecular Catalysis B: Enzymatic, 18 (2002), pp. 325-331). Fernandesa M L M et al. (Journal of Molecular Catalysis B. Enzymatic, 30 (1) 2004, pp. 43-49) describes hydrolysis and synthesis reactions catalysed by TLL lipase in the AOT/Isooctane reversed micellar system. Bilyk et al. (Journal of American Oil Chemist's Society, 68 (1991), pp. 320-323) report 76% hydrolysis by use of fungal lipases in presence of secondary amines, at moderate temperatures within 20 hrs. Further improvements in the yields have also been reported at 45° C.
Kulkarni et al. (Indian Journal of Biotechnology, 4 (2005), pp. 241-245) report optimization of enzymatic hydrolysis of castor oil with reference to reactor and reaction conditions. Ramachandran et al. (Biochemical Engineering Journal, 34 (2007), pp. 228-235) describes use of packed bed reactors with immobilized lipases for studying kinetics of hydrolysis of different oils and for improving the operational stability of lipases used in hydrolysis reactions. Goswami et al. (Bioresource technology, 101 (1) 2010, pp. 6-13) describes surfactant enhanced hydrolysis of castor oils for production of fatty acids. Martinez et al. describes hydrolysis of canola oil in a continuous flow of supercritical CO2 through a packed-bed reactor (Biocatalysis and Biotransformation, 12 (2) 2002, pp. 147-157). Helena Sovova et al. describes hydrolysis of blackcurrant seed oil catalysed by Lipozyme in a packed-bed reactor using supercritical CO2 (Chemical Engineering Science, 58 (11) 2003, pp. 2339-2350).
WO 91/016442 and U.S. Pat. No. 5,116,745 describe a process for the selective hydrolysis of triglycerides to 2-acyl glycerides. The process uses a primary lower alkyl alcohol, an aqueous buffer system and a 1,3-lipase. The 2-acyl monoglycerides can be used to make stereospecific 1,2-diacyl glycerides or 2,3-diacyl glycerides through esterification with acid anhydrides and 1,3-lipase catalysis. Stereospecific triglycerides can be made from these materials by standard esterification reactions under conditions which control rearrangement.
WO90/013656 describes a two-step enzymatic process involving lipase-catalyzed transesterification of triglycerides followed by low-temperature crystallization for preparing oil based products significantly enriched in omega-3 fatty acids. The process yields a mixture of highly pure monoglycerides, at least 60% of which contain omega-3 fatty acids. WO 90/04033 describes a process for the production of high purity monoglycerides by lipase-catalyzed transesterification. The method described comprises combining oils or pure triglycerides with alcohol, a small amount of water and a lipase. The reaction proceeds under mild conditions, and produces high yields of beta-monoglyceride product.
U.S. Pat. No. 6,500,974 describes a process for the preparation of a monoglyceride by reacting a fatty acid and glycerol in the presence of a food grade polar solvent and avoiding the use of catalysts. Eitel Pastor et al. (Biocatalysis and Biotransformation, 12 (2) 1995, pp. 147-157) describes direct esterification of glycerol with stearic acid or transesterification using ethyl stearate as acyl donor in the presence of Candida antarctica lipase (Novozym-435) using a variety of solvents of differing polarity.
In almost all cases, the hydrolysis was either incomplete or required longer reaction time which is more than three days to achieve completion. The effectiveness of lipases as catalysts is often offset by the high costs of production and isolation so that research groups are constantly striving to increase the yields of enzymes or productivity of enzymes. Further, commercial applications have been limited by high enzyme consumption, long reaction times and low productivity that have impeded successful industrial application.
Typically, lipase catalyzed enzymatic hydrolysis has been carried out using oil in water or water in oil emulsions where, the reusability of enzyme solution poses a problem if enzyme is used in free form. Also, immobilized enzyme preparations suffer from substrate accessibility issues wherein poor diffusibility of substrate in a non-homogeneous media restricts its efficient conversion.
None of the methods of the prior art provides the three desirable attributes namely, low cost of enzyme catalyst, complete hydrolysis of the oil and high enzyme stability. In the cited prior arts, no attempts have been made to separate the incompletely hydrolyzed oils (MAGs and DAGs) and FAs.
All the reports on enzymatic monoglyceride synthesis is primarily focused on the glycerolysis of various substrates like castor oil, soybean oil, coconut oil, palm oil, rapeseed oil, rice bran using glycerol. MAG production via glycerolysis using different oils and glycerol is an expensive process.
Therefore, there is a need to develop an efficient process for production of oleochemicals such as fatty acids and glycerol from oils. The process may be a process of hydrolysis of oils and/or fats, which bypasses the glycerol mediated hydrolysis i.e. glycerolysis and results in higher of fatty acids, MAGs directly through controlled oil hydrolysis.
Additional aspects of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practicing the invention. The invention is set forth and particularly pointed out in the appended claims, and the present disclosure should not be construed as limiting the scope of the claims in any way. The following detailed description includes exemplary representations of various embodiments of the invention, which are not restrictive of the invention, as claimed. The accompanying figures constitute a part of this specification and, together with the description, serve only to illustrate various embodiments and not limit the invention.
Citation of various references in this application, is not an admission that these references are prior art to the invention.
None of the enzymatic hydrolysis processes disclosed in the art describe the formation of a homogenous mixture of oil and water. Additionally, the processes are extremely time consuming and the hydrolysis takes up to 72 hours for completion. Hence there is a need in the art for a quick and easier process for enzymatic hydrolysis of fats and oils in a homogenous mixture.
The present invention provides an enzyme catalyzed process for the hydrolysis of fats, oils and combinations of fats and oils which can be completed in under 6 hours.